The research proposed here is an investigation of the molecular organization of naturally occuring, gene-sized DNA segments from the macronucleus of a protozoan, Oxytricha. These small DNA peices (about 1 micron long) are produced during the development of macronucleus from a micronucleus, a process which brings about not only a segmentation of the genome, but also the elimination of about 90% of the DNA sequences. The macronuclear genome, therefore, consists of short DNA segments with relatively simple sequence complexity. The macronucleus carries on all nuclear metabolism in vegetative cells. Therefore, these DNA pieces must contain all of the genes required for vegetative growth and all the sequences necessary to control the transcription of these genes. This research takes advantage of these unique properties of macronuclear DNA to ask questions about the structure, transcription and replication of eukaryotic genes. First, how much of a macronuclear DNA piece is transcribed: are there non-transcribed control regions and what is their approximate size? Experimentally, this involves analysis of molecular hybrids between entact macronuclear DNA pieces and the RNAs for which they code, using hydroxyapatite chromatography and S1 nuclease digestion. Second, where are non-transcribed regions located within a DNA segment? This can be examined by electron microscopy of the DNA-RNA hybrids. Third, do achromosomal DNA pieces replicate uniformly or differentially? This can be determined by hybridization of specific RNAs to macronuclear and micronuclear DNA. The RNAs used for these experiments will include rRNA, 5S RNA, and RNA complementary to macronuclear DNA segments linked to bacterial plasmids. The results of these experiments will give us a better understanding of the molecular mechanisms underlying the control of gene expression, and clearer insights into the organization of genes in chromosomes.